A high-molecular-mass neutral endopeptidase-24.5 from human lung

A high-Mr neutral endopeptidase-24.5 (NE) that cleaved bradykinin at the Phe5-Ser6 bond was purified to apparent homogeneity from human lung by (NH4)2SO4 fractionation, ion-exchange chromatography and gel filtration. The final enzyme preparation produced a single enzymically active protein band after electrophoresis on a 5% polyacrylamide gel. Human lung NE had an Mr of 650,000 under non-denaturing conditions, but after denaturation and electrophoresis on an SDS/polyacrylamide gel NE dissociated into several lower-Mr components (Mr 21,000-32,000) and into two minor components (Mr approx. 66,000). The enzyme activity was routinely assayed with the artificial substrate Z-Gly-Gly-Leu-Nan (where Z- and -Nan represent benzyloxycarbonyl- and p-nitroanilide respectively). NE activity was enhanced slightly by reducing agents, greatly diminished by thiol-group inhibitors and unchanged by serine-proteinase inhibitors. Human lung NE was inhibited by the univalent cations Na+ and K+. No metal ions were essential for activity, but the heavy-metal ions Cu2+, Hg2+ and Zn2+ were potent inhibitors. With the substrate Z-Gly-Gly-Leu-Nan a broad pH optimum from pH 7.0 to pH 7.6 was observed, and a Michaelis constant value of 1.0 mM was obtained. When Z-Gly-Gly-Leu-Nap (where -Nap represents 2-naphthylamide) was substituted for the above substrate, no NE-catalysed hydrolysis occurred, but Z-Leu-Leu-Glu-Nap was readily hydrolysed by NE. In addition, NE hydrolysed Z-Gly-Gly-Arg-Nap rapidly, but at pH 9.8 rather than in the neutral range. Although human lung NE was stimulated by SDS, the extent of stimulation was not appreciable as compared with the extent of SDS stimulation of NE from other sources.

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Fish skeletal muscle contains a novel serine proteinase with an unusual subunit composition

Biochemical Journal ◽

10.1042/bj2630471 ◽

1989 ◽

Vol 263 (2) ◽

pp. 471-475 ◽

Cited By ~ 18

Author(s):

E J E Folco ◽

L Busconi ◽

C B Martone ◽

J J Sánchez

Keyword(s):

Skeletal Muscle ◽

Proteinase Inhibitors ◽

Gel Filtration ◽

Serine Proteinase ◽

Molar Ratio ◽

Ph Optimum ◽

White Croaker ◽

Apparent Homogeneity ◽

Lysine Residues ◽

Sepharose 4B

Proteinase I, an enzyme previously shown to be able to degrade contractile and cytoskeletal elements of white-croaker (Micropogon opercularis) myofibrils, was purified to apparent homogeneity by chromatography on DEAE-Sephacel, octyl-Sepharose CL 4B and arginine-Sepharose 4B. Its Mr was determined to be 269,000 by Sephacryl S-300 gel filtration. Under denaturing conditions, the enzyme dissociated into two subunits with Mr 20,000 and 15,500, in a molar ratio of 1.8:1. Proteinase I showed a pH optimum of 8.5. The enzyme was strongly inhibited by several serine proteinase inhibitors, whereas inhibitors of the other types of proteinases did not affect, or only scarcely affected, its activity. Several N-terminal-blocked 4-methyl-7-coumarylamide substrates having either arginine or lysine residues adjacent to the fluorogenic group were efficiently hydrolysed by the enzyme. These results indicate that proteinase I is a trypsin-like serine proteinase. The enzyme appears to be distinct from other proteinases previously described in skeletal muscle, and might be involved in the catabolism of myofibrillar proteins.

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Isolation and characterization of cathepsin D from human gastric mucosa

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19813302 ◽

1981 ◽

Vol 46 (13) ◽

pp. 3302-3313 ◽

Cited By ~ 14

Author(s):

Jan Pohl ◽

Ladislav Bureš ◽

Karel Slavík

Keyword(s):

Gastric Mucosa ◽

Cathepsin D ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Exchange Chromatography ◽

Polyacrylamide Gel ◽

Human Gastric Mucosa ◽

Ph Optimum ◽

Isolation And Characterization

The molecular weight of the enzyme, purified by ion-exchange chromatography and affinity chromatography, was determined by gel filtration on Sephadex G-100 as 49 000. After treatment with 2-mercaptoethanol, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate resolved the enzyme into two chains, of molecular weights 33 000 and 18 000. This shows that in the native state the enzyme is composed of one light and one heavy chain. Isoelectric focusing in polyacrylamide gel gave four bands, the isoelectric points being 5.5, 6.1, 6.5 and 7.1. The optimum protein substrate (pH optimum 3.2-3.6) was haemoglobin. The best synthetic substrate was methyl ester of pyroglutamyl-histidyl-phenylalanyl-phenylalanyl-alanyl-leucine. The protease was inhibited by the inhibitor of cathepsin D from the potato tubers. It is concluded that the enzyme is cathepsin D from gastric mucosa.

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Characterization of a chelator-resistant proteinase from Thermus strain Rt4A2

Biochemical Journal ◽

10.1042/bj2950463 ◽

1993 ◽

Vol 295 (2) ◽

pp. 463-469 ◽

Cited By ~ 17

Author(s):

S A Freeman ◽

K Peek ◽

M Prescott ◽

R Daniel

Keyword(s):

Specific Activity ◽

Sequence Similarity ◽

Substrate Inhibition ◽

Gel Filtration ◽

Serine Proteinase ◽

Fold Increase ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Ph Optimum ◽

High Sequence Similarity

The Thermus isolate Rt4A2 was found to produce an extracellular chelator-resistant proteinase. The proteinase was purified to homogeneity by (NH4)2SO4 precipitation, cation-exchange chromatography, gel-filtration chromatography, and weak anion-exchange chromatography. The Rt4A2 proteinase was found to have properties typical of an alkaline serine proteinase. It had a pH optimum of 9.0 and was specifically inhibited by phenylmethanesulphonyl fluoride. Its isoelectric point was greater than 10.25. Its molecular-mass was 31.6 kDa as determined by SDS/PAGE. N-terminal sequencing has shown it to have high sequence similarity with other serine proteinases from Thermus species. The proteinase hydrolysed a number of substrates including fibrin, casein, haemoglobin, collagen, albumin and the synthetic chromogenic peptide substrate Suc-Ala-Ala-Pro-Phe-NH-Np. The specific activity of the purified proteinase using azocasein as substrate was 313 units/mg. Substrate inhibition was observed above an azocasein concentration of 0.05% (w/v). Esterase activity was directed mainly towards those substrates containing the aliphatic or aromatic residues of alanine, glycine, tryptophan, tyrosine and phenylalanine. Thermostability half-lives of greater than 7 days at 70 degrees C, 43 h at 80 degrees C and 90 min at 90 degrees C were found in the presence of 5 mM CaCl2. At 90 degrees C increasing the CaCl2 concentration 100-fold (0.5 mM to 50 mM) caused a 4.3-fold increase in the half-life of the enzyme from 30 to 130 min. Half-lives of 19.4 min at 100 degrees C and 4.4 min at 105 degrees C were found in the presence of 50 mM CaCl2. The metal chelators EGTA and EDTA reduced the stability at higher temperatures but had no effect on the activity of the proteinase. Activity was not stimulated by common metal activators such as Ca2+, Mg2+ and Zn2+.

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Caldolase, a chelator-insensitive extracellular serine proteinase from a Thermus spp

Biochemical Journal ◽

10.1042/bj2620409 ◽

1989 ◽

Vol 262 (2) ◽

pp. 409-416 ◽

Cited By ~ 16

Author(s):

G A Saravani ◽

D A Cowan ◽

R M Daniel ◽

H W Morgan

Keyword(s):

Specific Activity ◽

Gel Filtration ◽

Deae Cellulose ◽

Serine Proteinase ◽

Gel Permeation Chromatography ◽

Exchange Chromatography ◽

Acetate Buffer ◽

Ph Optimum ◽

Low Concentrations ◽

Low Ionic Strength

An extracellular alkaline serine proteinase from Thermus strain ToK3 was isolated and purified to homogeneity by (NH4)2SO4 precipitation followed by ion-exchange chromatography on DEAE-cellulose and QAE-Sephadex, affinity chromatography on N alpha-benzyloxycarbonyl-D-phenylalanyl-triethylenetetraminyl-Sepha rose 4B and gel-filtration chromatography on Sephadex G-75. The purified enzyme had a pI of 8.9 and an Mr determined by gel-permeation chromatography of 25,000. The specific activity was about 37,700 proteolytic units/mg with casein as substrate, and the pH optimum was 9.5. Proteolytic activity was inhibited by low concentrations of di-isopropyl phosphorofluoridate and phenylmethanesulphonyl fluoride, but was unaffected by EDTA, EGTA, o-phenanthroline, N-ethyl-5-phenylisoxazolium-3′-sulphonate, N alpha-p-tosyl-L-phenylalanylchloromethane, N alpha-p-tosyl-L-lysylchloromethane, trypsin inhibitors and pepstatin A. The enzyme contained approx. 10% carbohydrate and four disulphide bonds. No Ca2+, Zn2+ or free thiol groups were detected. It hydrolysed several native and dye-linked proteins and synthetic chromogenic peptides and esters. The enzyme was very thermostable (half-life values were 840 min at 80 degrees C, 45 min at 90 degrees C and 5 min at 100 degrees C). The enzyme was unstable at low ionic strength: after 60 min at 75 degrees C in 0.1 M-Tris/acetate buffer, pH 8, only 20% activity remained, compared with no loss in 0.1 M-Tris/acetate buffer, pH 8, containing 0.4 M-NaCl.

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Purification and characterization of cysteine-S-conjugate N-acetyltransferase from pig kidney

Biochemical Journal ◽

10.1042/bj3170213 ◽

1996 ◽

Vol 317 (1) ◽

pp. 213-218 ◽

Cited By ~ 13

Author(s):

Achim AIGNER ◽

Martina JÄGER ◽

Ralf PASTERNACK ◽

Peter WEBER ◽

Dirk WIENKE ◽

...

Keyword(s):

Proteinase Inhibitors ◽

Cysteine Proteinase ◽

Gel Filtration ◽

Protein Band ◽

Exchange Chromatography ◽

Polyclonal Antiserum ◽

Partial Purification ◽

Ph Optimum ◽

Sds Page

Microsomal cysteine-S-conjugate N-acetyltransferase catalyses the N-acetylation of various S-substituted cysteines in liver and kidney. We describe here the purification and more detailed characterization of this enzyme catalysing the final reaction of mercapturic acid biosynthesis, and thus playing a crucial role in the detoxicating metabolism of many xenobiotics. The solubilization of cysteine-S-conjugate N-acetyltransferase by deoxy-BIGCHAP [N,N´-bis-(3-d-gluconamidopropyl)deoxycholamide] was the prerequisite for partial purification by means of anion-exchange chromatography. The molecular mass of the enzyme was determined by gel filtration. A polyclonal antiserum was raised against the excised protein band from SDS/PAGE and purified antibodies were used for the complete purification of native cysteine-S-conjugate N-acetyltransferase by immunoaffinity chromatography. A dimeric form of the enzyme was sometimes detected on SDS/PAGE, depending on the degree of purification. For further characterization of cysteine-S-conjugate N-acetyltransferase, the stability of catalytic activity, the pH optimum and Km values were determined. The inhibitory effects of various agents were tested, revealing a substantial, yet not complete, loss of cysteine-S-conjugate N-acetyltransferase activity after treatment with cysteine proteinase inhibitors or probenecid under various conditions.

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Purification and Characterization of an X-Prolyl-Dipeptidyl Peptidase from Lactobacillus sakei

Applied and Environmental Microbiology ◽

10.1128/aem.67.4.1815-1820.2001 ◽

2001 ◽

Vol 67 (4) ◽

pp. 1815-1820 ◽

Cited By ~ 38

Author(s):

Yolanda Sanz ◽

Fidel Toldrá

Keyword(s):

Proteinase Inhibitors ◽

Divalent Cations ◽

Gel Filtration ◽

Serine Proteinase ◽

Anion Exchange Chromatography ◽

Dipeptidyl Peptidase ◽

Exchange Chromatography ◽

Lactobacillus Sakei ◽

N Terminus ◽

Hydrolysis Of

ABSTRACT An X-prolyl-dipeptidyl peptidase has been purified fromLactobacillus sakei by ammonium sulfate fractionation and five chromatographic steps, which included hydrophobic interaction, anion-exchange chromatography, and gel filtration chromatography. This procedure resulted in a recovery yield of 7% and an increase in specificity of 737-fold. The enzyme appeared to be a dimer with a subunit molecular mass of approximately 88 kDa. Optimal activity was shown at pH 7.5 and 55°C. The enzyme was inhibited by serine proteinase inhibitors and several divalent cations (Cu2+, Hg2+, and Zn2+). The enzyme almost exclusively hydrolyzed X-Pro from the N terminus of each peptide as well as fluorescent and colorimetric substrates; it also hydrolyzed X-Ala at the N terminus, albeit at lower rates. Km s for Gly-Pro- and Lys-Ala-7-amido-4-methylcoumarin were 29 and 88 μM, respectively; those for Gly-Pro- and Ala-Pro-p-nitroanilide were 192 and 50 μM, respectively. Among peptides, β-casomorphin 1-3 was hydrolyzed at the highest rates, while the relative hydrolysis of the other tested peptides was only 1 to 12%. The potential role of the purified enzyme in the proteolytic pathway by catalyzing the hydrolysis of peptide bonds involving proline is discussed.

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Purification and characterization of a chymosinlike protease from the gastric mucosa of harp seal (Pagophilus groenlandicus)

Canadian Journal of Biochemistry and Cell Biology ◽

10.1139/o84-091 ◽

1984 ◽

Vol 62 (8) ◽

pp. 699-708 ◽

Cited By ~ 27

Author(s):

K. Shamsuzzaman ◽

N. F. Haard

Keyword(s):

Gastric Mucosa ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Exchange Chromatography ◽

Polyacrylamide Gel ◽

Ph Optimum ◽

Porcine Pepsin ◽

Milk Clotting

Four zymogens of acidic proteases A, B, C, and D were isolated from the gastric mucosa of harp seals by ion-exchange chromatography on a diethylaminoethyl-Sephadex A-50 column. The major zymogens were A and C, and the ratio of zymogen A to zymogen C was greater in extracts from 1-week-old animals than in extracts from adult animals. Zymogens A and C were further purified by affinity chromatography using carbobenzoxy-D-phenylalaninetriethylene tetramine Sepharose and gel filtration on a Sephadex G-100 column. Certain physical and catalytic properties of proteases A and C were compared with those of calf chymosin (EC 3.4.23.4) and porcine pepsin (EC 3.4.23.1). Zymogen C and the corresponding enzyme were homogeneous on analytical polyacrylamide gel electrophoresis. Zymogen A was homogeneous as judged by sodium dodecyl sulphate (SDS) – polyacrylamide gel electrophoresis and high performance liquid chromatography, but was heterogenous by polyacrylamide gel electrophoresis at pH 8.3. Zymogens A and C had molecular weights of 33 800 and 44 000, respectively, as estimated by SDS–polyacrylamide gel electrophoresis. Protease A had an isoelectric point of 4.90. Protease A was similar to calf chymosin with respect to several criteria. It had a higher ratio of milk-clotting to proteolytic activity than those of seal protease C and porcine pepsin and had a pH optimum of 2.2–3.5 for hemoglobin hydrolysis. It did not inactivate ribonuclease, had very low activity on N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine and lost activity in 6 M urea. These results indicate protease A is chymosinlike.

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Purification and characterization of prostate-specific antigen (PSA) complexed to alpha 1-antichymotrypsin: potential reference material for international standardization of PSA immunoassays

Clinical Chemistry ◽

10.1093/clinchem/41.9.1273 ◽

1995 ◽

Vol 41 (9) ◽

pp. 1273-1282 ◽

Cited By ~ 42

Author(s):

Z Chen ◽

A Prestigiacomo ◽

T A Stamey

Keyword(s):

Molecular Mass ◽

Prostate Specific Antigen ◽

Specific Antigen ◽

Gel Filtration ◽

Exchange Chromatography ◽

Molar Ratio ◽

Free Psa ◽

Apparent Homogeneity ◽

International Standardization

Abstract We describe for the first time a protocol to purify to apparent homogeneity an in vitro-prepared complex of prostate-specific antigen (PSA) and alpha 1-antichymotrypsin (ACT) by using a combination of gel filtration and ion-exchange chromatography. The purity of the PSA-ACT complex was confirmed by gel electrophoresis and Western blot. The PSA-ACT complex was stable in the pH range 6.0 to 7.8; it was also stable in various matrices, temperatures, and high concentrations of salt. Purification of the PSA-ACT complex was highly reproducible. An absorptivity of 0.99 L x g-1 x cm-1 at 280 nm was assigned to the PSA-ACT complex, based on amino acid analysis. Because PSA and ACT bind in a 1:1 molar ratio, we determined the molecular mass of the PSA-ACT complex as the mass encoded by the cDNA of ACT (plus 26% carbohydrate) plus the molecular mass of PSA (28,430 Da), which totals 89,280 Da. Using this material, we made two common calibrators, one of 100% PSA-ACT complex and one of 90% PSA-ACT complex plus 10% free PSA by volume (90:10 calibrator). Substitution of these calibrators for the manufacturers' calibrators in nine commercial immunoassays substantially reduced differences between immunoassays, especially for serum PSA values between 4 and 10 micrograms/L. The 90:10 calibrator is recommended as a universal calibrator for international standardization of PSA immunoassays.

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Isolation and amino-acid sequence of two inhibitors of serine proteinases, members of the squash inhibitor family, from Echinocystis lobata seeds.

Acta Biochimica Polonica ◽

10.18388/abp.1996_4484 ◽

1996 ◽

Vol 43 (3) ◽

pp. 507-513 ◽

Cited By ~ 3

Author(s):

D Stachowiak ◽

A Polanowski ◽

G Bieniarz ◽

T Wilusz

Keyword(s):

Amino Acid ◽

Proteinase Inhibitors ◽

Sequence Similarity ◽

Serine Proteinase ◽

Exchange Chromatography ◽

Association Constants ◽

Cathepsin G ◽

Amino Acid Residues ◽

Mature Seeds ◽

Echinocystis Lobata

Two serine proteinase inhibitors (ELTI I and ELTI II) have been isolated from mature seeds of Echinocystis lobata by ammonium sulfate fractionation, methanol precipitation, ion exchange chromatography, affinity chromatography on immobilized anhydrotrypsin and HPLC. ELTI I and ELTI II consist of 33 and 29 amino-acid residues, respectively. The primary structures of these inhibitors are as follows: ELTI I KEEQRVCPRILMRCKRDSDCLAQCTCQQSGFCG ELTI II RVCPRILMRCKRDSDCLAQCTCQQSGFCG The inhibitors show sequence similarity with the squash inhibitor family. ELTI I differs from ELTI II only by the presence of the NH2-terminal tetrapeptide Lys-Glu-Glu-Gln. The association constants (Ka) of ELTI I and ELTI II with bovine-trypsin were determined to be 6.6 x 10(10) M-1, and 3.1 x 10(11) M-1, whereas the association constants of these inhibitors with cathepsin G were 1.2 x 10(7) M-1, and 1.1 x 10(7) M-1, respectively.

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Optimization of Metal Ions in Sugarcane Bagasse Fermenting Medium for the Production of Streptokinase by Streptococcus equisimilis

Science Letters ◽

10.47262/sl/9.2.132021003 ◽

2021 ◽

Vol 9 (2) ◽

pp. 24-30

Keyword(s):

Metal Ions ◽

Sugarcane Bagasse ◽

Specific Activity ◽

Gel Filtration ◽

Value Added ◽

Cost Effective ◽

Fermentation Medium ◽

Exchange Chromatography ◽

Sds Page ◽

The Cost

Streptokinase is a fibrinolytic enzyme and a product of β-hemolytic Streptococci strains. This enzyme is used as a medication to break down clots in some cases of heart disease. Streptococcus equisimilis, a species of group C Streptococci, is widely used for the production of streptokinase by fermentation technology. In this study, the sugarcane bagasse fermentation medium was optimized for metal ions (KH2PO4, MgSO4.7H2O, CaCO3 and NaHCO3) at various levels to attain the maximal production of streptokinase. Sugarcane bagasse was used due to its profuse availability and as an ideal substrate for microbial processes for the manufacturing of value-added products. The results showed that maximal streptokinase production was found at 0.04% KH2PO4, 0.04% MgSO4.7H2O, 0.15% NaHCO3 and 0.04% CaCO3. Finally, the optimized medium resulted in 84.75 U/mg specific activity and 74.5% recovery. The purification process was carried out simultaneously using ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. Finally, a purified sample of streptokinase was run on SDS-PAGE and resolute 47 kDa molecular weight. The use of β-hemolytic Streptococci to obtain streptokinase is not free from health risks and is related to anaphylaxis. This study provides a way forward for the cost-effective ways to obtain streptokinase for the treatment of thrombosis.

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